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Leukotriene (LT) C4 synthase is an integral membrane protein that catalyzes the conjugation of LTA4 to reduced glutathione to form LTC4. LTC4 synthase has been cloned and characterized from transformed cell lines, but the protein has not been defined from a tissue source. LTC4 synthase was purified to homogeneity from human lung tissue, utilizing S-hexyl glutathione chromatography followed by LTC4 affinity chromatography. A greater than 100,000-fold purification with a yield of 8 to 25% (n = 4) was achieved. The purified LTC4 synthase migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as an 18-kD protein, and its 19 N-terminal amino acid sequence is identical to that of purified LTC4 synthase from KG-1 myeloid cells or from expression cloning of a KG-1 library in COS cells. Using a rabbit polyclonal IgG raised against purified LTC4 synthase, SDS-PAGE immunoblotting of LTC4 synthase from human lung tissue, eosinophils, KG-1 cells, and platelets showed an 18-kD protein. Immunofluorescence staining of alveolar macrophages in human lung sections with the anti-LTC4 synthase IgG revealed LTC4 synthase to be largely perinuclear in distribution. Thus, LTC4 synthase, the biosynthetic enzyme responsible for the formation of cysteinyl LTs, is present in lung tissue in a form apparently identical to that of hematopoietic cells.

1 and its active metabolites, LTD 4 and LTE 4 , are the major components of the biologic activity previously known as the slow reacting substance of anaphylaxis. When inhaled, these arachidonic acid-derived lipid mediators exert profound smooth muscle constrictor effects on the airways of individuals with and without asthma (1, 2). The cysteinyl leukotrienes are further implicated in the pathogenesis of asthma by the presence of their metabolites in the urine of patients with acute severe asthma (3). Moreover, cysteinyl leukotriene synthesis inhibitors or receptor antagonists significantly ameliorate the persistent pulmonary function abnormalities of individuals with asthma (4) and the exacerbations of bronchial asthma elicited by exercise (5), inhalation of specific allergens (6), and the idiosyncratic response to aspirin (7,8).The formation of the cysteinyl leukotrienes is initiated by transmembrane stimuli that increase the levels of intracellular calcium, leading to the translocation of cytosolic phospholipase A 2 (9) and 5-lipoxygenase to the perinuclear membrane (10). Cytosolic phospholipase A 2 liberates arachidonic acid from phospholipids (11) for presentation to 5-lipoxygenase by 5-lipoxygenase activating protein (FLAP) (12, 13), an integral perinuclear membrane protein (10). 5-Lipoxygenase catalyzes the sequential formation of 5-hydroperoxyeicosatetraenoic acid and the unstable epoxide, LTA 4 (14, 15). LTC 4 synthase then catalyzes the conjugation of LTA 4 with reduced GSH to form intracellular LTC 4 (16, 17). LTC 4 synthase is an 18-kDa integral membrane protein, which has been localized to the perinuclear region of alveolar macrophages (18) and recognized either by enzymatic function and/or by SDS-polyacrylamide gel electrophoresis immunoblot analysis in some hematopoietic cell populations such as eosinophils, basophils, mast cells, and platelets (18 -21). After carrier-mediated export of LTC 4 (22), the GSH adduct is cleaved sequentially by ␥-glutamyl transpeptidase to form LTD 4 (23) and by dipeptidases to yield LTE 4 (24), both of which are biologically active metabolites.The cDNA and consensus amino acid sequence of LTC 4 synthase bear no homology to that of any member of the GSH S-transferase family, but instead, the deduced amino acid sequence shows significant homology to the amino acid sequence of FLAP (25,26). The predicted secondary structure of LTC 4 synthase contains three hydrophobic domains and two hydrophilic loops, which align identically with the predicted secondary structure of FLAP (25). A sequence of 22 amino acid residues at the carboxyl terminus of the first hydrophilic loop of FLAP is believed to bind the released arachidonic acid and is the site at which FLAP inhibitors act to prevent cellular 5-li-